The present invention relates generally to procedures and systems for immunoabsorbent assay. More particularly, the present invention relates to a microassay rod and card system which is capable of testing a single small sample simultaneously for the presence of numerous different biologically important substances.
Immunoassay systems are all based upon the specific yet reversible chemical reactions which occur between an antigen and a corresponding antibody. In general, a known antigen or antibody is absorbed onto or otherwise attached to a solid support. The fixed antigen or antibody is then used to screen or probe various solutions of interest to detect the presence of antibodies or antigens which are immunoreactive with the bound antigen or antibody.
For example, if a test solution is to be screened or probed for a particular antibody, a known antigen which combines specifically with the antibody to be tested for is attached to a suitable support structure. The fixed known antigen is then used to probe a test solution for the presence of the antibody. If the specific antibody is present, it will combine with the fixed antigen to thereby become indirectly bound to the fixed support material. Any unbound antibody and other interfering substances are then removed from the support surface by washing. The presence of any antibody indirectly bound to the fixed support by way of the known antigen is then detected by the use of a second antibody which has a particular characteristic, or includes a suitable marker which can be measured.
Many different types of antibody markers have been developed for use in immunoassay systems. Antibodies tagged with radioisotopes have been popular markers for use in radioimmunoassay; however, the use of radioisotopes requires expensive specialized equipment and specialized handling features. Enzyme Linked Immunoabsorbent Assay (ELISA) is another immunoassay system which has experienced widespread popularity. ELISA is based upon the use of an enzyme linked antibody marker to detect the presence of antibody bound to the fixed antigen. Typically, an enzyme such as horseradish peroxidase is linked to an immunoglobulin which is capable of combining with the antibody being tested. The enzyme is capable of catalyzing an easily measured color producing reaction. The popularity of ELISA type procedures is based in large part upon the ease with which the colored product produced by the enzyme-linked antibody can be detected. In addition to radioisotope and enzyme linked antibody markers, fluorescent compounds, such as fluorescein have been used to provide a fluorescent antibody marker.
With the advent of monoclonal antibodies, and highly specific antigens, it is now possible using immunoassay techniques to probe various test solutions such as blood, sputum or urine specifically and selectively for numerous different immunochemically reactive substances, i.e., those substances capable of engaging in antibody-antigen type reactions. It would be desirable to use these newly available immunochemical probes to provide an assay system in which a large number of specific antibodies or antigens could be screened for simultaneously using a single aliquot of sample and a single test system.
For the most part, the present immunoassay systems utilize a single solid support coated with a single known antigen or antibody. The solid support is then used to probe one or more samples for a single immunoreactive substance (i.e., antigen or antibody). When screening a test solution for more than one antigen or antibody, the test solution must be separated to provide separate aliquots for testing. This type of screening procedure is especially undesirable where a large number of antigens or antibodies are being tested for and where the amount of test solution available is small.
In 1982, Hawkes, et al. published a paper describing a Dot-Immuno-binding Assay (Hawkes, R., Niday, E., and Gordon, J., Analytical Biochemistry 119, 142-147 (1982) which provides for the simultaneous screening of a test solution for a number of antibodies. The Dot-Imnunobinding Assay basically involves dotting a number of different antigens in different places on the surface of a nitrocellulose filter paper. Upon drying, the antigens are bound non-covalently to the paper and remain attached to it during subsequent manipulations. This single piece of filter paper is then used as the solid support to simultaneously test for the presence of antibodies reactive with the dotted antigens. An enzyme-linked antibody and associated chromogenic substrate for the enzyme is then used to "develop" the filter paper to detect the presence of antibodies bound to the dotted antigens on the filter paper.
The Dot-Immunobinding Assay system is well suited for screening a test solution for a relatively small number of antigen or antibodies. Difficulties arise, however, when a large number of antigens or antibodies are to be tested. Such difficulties include problems in spotting a relatively large number of well defined (i.e., non-overlapping) dots of known antigens or antibody on a single filter paper. Problems with reading the closely spaced dots are also expected. If the dots are spaced apart on the filter paper to maintain dot integrity, it is then necessary to use an inordinately large sample of test solution to conduct a simultaneous analysis since a relatively large area of filter paper must be treated with the solution to be tested. Accordingly, there has been a continuing need to provide a multiple immunoassay system which can conveniently, quickly, efficiently and accurately screen a microsized sample for a large number of antigens and/or antibodies.
Although the radioisotope markers, enzyme linked markers and fluorescent markers are all well suited for immuno-absorbent assay of individual antibodies, it would be desirable to provide a new marker or tag which can be used in a multiple immunoassay system to provide accurate and quick identification of a large number of discrete bound antibodies. It would also be desirable to provide a marker whose characteristic can be easily measured and interfaced with computer processing equipment.
It would also be desirable to provide a microassay rod in which a position marker or some other means is provided which allows determination of the absolute position of the microassay rod as it is passed through a reading device. The provision of such markers would facilitate the identification of each probe on the individual rod and insure accurate determinations. In addition, it would be desirable to provide a method in which the detection and measurement of substances of interest which are bound to the various microassay rod probes is maximized.